Cyclic GMP mediates vascular smooth muscle relaxation to nitric oxide and atrial natriuretic peptides. Endogenous vascular tone, tissue perfusion, and the growth and differentiation state of vascular smooth muscle are all regulated by this important second messenger. The receptor protein for cGMP in vascular smooth muscle cells is the protein serine/threonine kinase, cGMP-dependent protein kinase (cGMP kinase), but little is known concerning the mechanisms by which this kinase regulates vascular smooth muscle fell function. At least one mechanism by which cGMP kinase evokes relaxation is by activating cellular events that lead to the reduction in intracellular Ca2+ levels. It has recently been shown that cGMP kinase is localized to the sarcoplasmic reticulum of rat aortic vascular smooth muscle cells, and catalyzes the phosphorylation of proteins that regulate the sequestration of Ca2+ into the reticulum. Other mechanisms exist for cGMP-dependent reduction of intracellular Ca2+, including the inhibition of phospholipase C activity and the stimulation of Ca2+-activated K+ channels, suggesting the kinase has a multiplicity of actions. In order for rapid and efficient cGMP-regulated protein phosphorylation to occur in VSM cells, the cGMP kinase must be localized to cellular structures (organelles) that contain these substrate proteins. One protein that may be involved in localizing cGMP kinase to substrates is the cytoskeletal intermediate filament protein vimentin. We propose to study the mechanism by which cGMP binds to cytoskeletal vimentin in vitro and in the intact cell. In specific aim #1, we will define the binding domains of cGMP kinase for the cytoskeletal protein vimentin using recombinant cGMP kinase molecules containing deletions in these binding domains. Binding studies with recombinant cGMP kinase and purified vimentin will be performed using different biochemical and biophysical methods. In specific aim #2, we will determine the importance of cGMP binding to cytoskeletal vimentin in the intact cell by studying the effects of deletions in the vimentin binding domains in vascular smooth muscle cells transfected with cDNAs encoding the recombinant cGMP kinases. The cellular responses to be studied include SR protein phosphorylation and intracellular Ca2 levels. Disruptions in cGMP kinase binding to vimentin may lead to disruptions in protein phosphorylation and Ca2+ regulation. Cytoskeletal proteins also interact with protein tyrosine kinase (PTK) signaling, especially in phospholipase C signaling. In the third specific aim, we will determine whether inhibitors of PTKs and protein tyrosine phosphatases (PTPs) affect cGMP kinase-mediated phospholipase C inhibition. Both activation and translocation to the plasma membrane will be examined. Our knowledge regarding the mechanism of action of cGMP kinase in vascular smooth muscle physiology is not complete. These new approaches towards investigating the role of this kinase in vascular smooth muscle cell function will increase our understanding of the mechanisms by which the nitric oxide- cGMP signaling pathway regulates vascular physiology. As a result, new avenues of investigation into the role of the cytoskeleton and protein tyrosine phosphorylation in cGMP action will be opened, and may allow us to understand more completely the mechanisms by which vascular function is altered in cardiovascular and other disorders.